Site-Directed Mutagenesis Protocol using PCR

  1. Design primer pair so that the mutation (s) are near the middle of the primer, and the primer has a melting temperature high enough
  2. Perform 12-16 rounds of PCR on whole vectors
    (Include a control sample where you just incubate with the primary one, the other primary displace water.)
  3. Check the PCR product on gel; if there is no product, lowering the annealing temperature PCR; if there is a smear, increasing the annealing temperature PCR
  4. Digest with DpnI for 2 hours at 37 degrees C.
  5. Transform into competent cells, and plate to plate agar.
  6. Select a colony and grow bacteria in a culture overnight.
  7. Purify plasmid, check the concentration and insert DNA sequences to ensure that the desired mutation had occurred, and no more random mutations are present.
  8. Express your protein of interest via transfection in mammalian cells or other methods.